Inflammation is a process well known for its implication in acute and chronic diseases and disorders in the biomedical field. Although inflammation is a natural process associated with cell and tissue defense and regeneration, disorganized inflammation can contribute to (or is implicated in) many processes that are harmful to cells and tissues.
Inflammation is the body's reaction to infectious agents, antigen challenge or physical, chemical or traumatic injury (Stvrtinova et al., 1995). The main purpose of inflammation is to bring fluids, proteins, and cells from the blood into the damaged tissues. The main features of the inflammatory response are (i) vasodilation (widening of the blood vessels to increase blood flow); (ii) increased vascular permeability that allows diffusible components to enter the tissues; (iii) cellular infiltration by chemotaxic, or directed movement of inflammatory cells through the walls of blood vessels into the site of injury; (iv) changes in biosynthetic, metabolic, and catabolic profiles of the affected tissues; and (v) activation of cells of the immune system as well as enzymatic systems of the blood plasma.
In general, the inflammation response is quite efficient in managing and repairing damages induced by injury or infectious agent. The degree to which these phenomena occur is normally proportional to the severity of the injury or the extent of the challenge. However, inflammation can become harmful to tissues when it develops in a disorganized, disproportionate or undesired manner and can lead to diseases and disorders.
The acute inflammation response is short lasting and involves all of the previously mentioned features of inflammation. Acute inflammation, when it proceeds in a disorganized fashion, can cause many harmful effects such as the digestion/destruction of normal tissues, excessive swelling that may lead to obstruction of blood flow, resulting in ischemia damage, hypersensitive reaction to non threatening entities (e.g. allergens), etc.
The chronic inflammation reaction may be seen as a long-lasting inflammation, where the inflammatory agent is continually present. In this context, chronic inflammation is essentially observed under conditions of delayed hypersensitivity. However, chronic inflammation may be seen is cases where the inflammatory agent is not continuously present, as is the case of in asthma, arthritis or inflammatory bowel disease, and it may also be related to neurological or genetic disorders. In this case, one or more inflammatory components contribute to the etiology and perpetuation of inflammation.
The process of inflammation is driven and modulated by a complex interplay between products of the plasma enzyme systems, lipid mediators (arachidonic acid metabolites such as prostaglandins and leukotrienes), vasoactive mediators released from inflammatory cells, and, in particular, cytokines.
Prostaglandins (derived from eicosanoic essential fatty acids) are produced during an inflammatory response by inflammation-related biochemical pathways and are responsible for mediating the clinical manifestations characteristic of inflammation. The major source for the production of inflammation-related prostaglandins is arachidonic acid. Arachidonic acid can be metabolized by one of two cyclo-oxygenases (COX-1 or COX-2) producing inflammatory metabolites. The increased production of pro-inflammatory metabolites in inflamed tissues is due to the specific up-regulation of COX-2 (Maier et al., 1990). The increased expression of COX-2 during an inflammatory response is believed to be induced (in part) by exposure to bacterial endotoxins and/or the release of pro-inflammatory cytokines (Isakson, 1995; Raz et al., 1989; O'Sullivan et al., 1992), although other materials may increase expression of COX-2 as well.
In contrast, COX-1 is constitutively expressed in most tissues and has been proposed to be involved in the maintenance of physiological functions such as platelet aggregation, cytoprotection in the stomach, and in part, the regulation of normal kidney function (Prasit et al., 1995; Pinto et al., 1995; Whittle et al., 1980).
In addition to the production of pro-inflammatory eicosanoid metabolites via the cyclo-oxygenase pathways, arachidonic acid also serves as the source for the production of another class of inflammation-related metabolites produced by a family of related enzymes called lipoxygenases (LOX). In particular, 5-LOX catalyzes the first step of a biochemical cascade that culminates in the biosynthesis of a class of molecules termed leukotrienes (Sirois, 1985). Leukotrienes have been implicated as important mediators of inflammatory responses, such as anaphylaxis, suggesting that potent inhibitors of 5-LOX would provide an approach to limit the deleterious effects of all the products of this pathway. Elevated 15-LOX activity has been associated with conditions such as asthma and hypereosinophilia. Selective inhibition of 5-, 12-, or 15-LOX may provide an agent with a definite therapeutic advantage.
In addition to prostaglandins and leukotrienes, cytokines also play a critical role in the inflammatory response. They are produced at the onset of inflammation development and are responsible for the eventual outcome of the inflammation process as well as its resolution. When injury or challenge occurs, cytokines are released from inflammatory cells (mast cells, basophils, endothelial cells, macrophages and neutrophils). The release of many different cytokines is activated during this process including the pro-inflammatory interleukins IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-α). In order to counteract an exaggerated inflammation, anti-inflammatory cytokines such as IL4, IL-10, IL-13, and transforming growth factor (TGF-β) are also produced. Although many cytokines are involved in the inflammation process, some cytokines have a central role in the process and have recently been examined as possible targets for anti-inflammatory products.
Acute and chronic inflammation is most often treated with compounds with anti-inflammatory activity. Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are among the most frequently used drugs currently available. Originally, the medicinal utility of classical NSAIDs was suspected to be due to their ability to inhibit the activities of COX-1 (Mitchell et al., 1993; Meade et al., 1993). Today, it is recognized that NSAIDs also have anti-inflammatory activity due to inhibition of COX-2 as well. Other biochemical activities associated with NSAIDs include inhibition of inflammatory mediators other than those mentioned above (i.e. histamine, serotonin, kinins), inhibition of oxidative phosphorylation, displacement of anti-inflammatory peptides from serum albumin, or displacement of peptides that hyperpolarize neuronal membranes in inflamed tissue (Foye, 1989).
The key roles played by arachidonic acid metabolites produced by COX-2 and 5-, 12-, 15-LOX in mediating inflammatory responses has prompted extensive research to identify compounds capable of specifically inhibiting the enzymatic activities of COX-2, 5-, 12-, 15-LOX, or more than one simultaneously (i.e., dual inhibitors). Compounds capable of inhibiting COX-2 (but not COX-1) and/or 5-LOX would be of great use as anti-inflammatory agents without the ensuing deleterious side effects common to most non-steroidal anti-inflammatory drugs. Alternatively, compounds inhibiting release of arachidonic acid or compounds antagonizing pro-inflammatory cytokines would be of potential therapeutic use, whether they are steroidal (SAID), non-steroidal. (NSAID), cytokine suppressive (CSAID) or other anti-inflammatory drugs. Such inhibitory compounds would have great clinical utility in the treatment of such conditions as pain, fever, asthma, allergic rhinitis, rheumatoid arthritis, osteoarthritis, gout, adult respiratory disease syndrome, inflammatory bowel disease, endotoxic shock, ischemia-induced myocardial injury, atherosclerosis, and brain damage caused by stroke. Such inhibitors could also be used topically for the treatment of acne, sunburn, psoriasis, eczema, and related conditions.
Though anti-inflammatory drugs are widely used to effectively treat inflammation, side effects of anti-inflammatory drug use such as steroid resistance, high doses, osteoporosis, catabolism of proteins and lipids, redistribution of lipidic masse, etc. are a major concern in medical research and drug development. One approach to alleviate side effects is to develop anti-inflammatory drug that have specific biochemical targets such as the development of NSAIDs that inhibit COX-2 (but not COX-1).
Although this strategy if current in terms of research and development of anti-inflammatory drugs, an alterative strategy would be to use current anti-inflammatory drugs in combination with a potentiation agent in order to heighten the efficacy of less effective anti-inflammatory drugs and, potentially, lower the dosage rate in order to alleviate some of the side effects.
Cytokines play a critical role in the inflammatory response. They are produced at the onset of inflammation development and are responsible for the eventual outcome of the inflammation process as well as its resolution. When injury or challenge occurs, cytokines are released from inflammatory cells (mast cells, basophils, endothelial cells, macrophages and neutrophils). The release of many different cytokines is activated during this process including the pro-inflammatory interleukins IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-α). In order to counteract an exaggerated inflammation, anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and transforming growth factor (TGF-β) are also produced.
Pro- to anti-inflammatory cytokines will determine the eventual outcome of inflammation by their relative proportions, their affinities, and their interactions. More accurately, an appropriate balance and interaction of pro- to anti-inflammatory cytokines will modulate the inflammation process in order to deal with the injury or challenge in the most efficient manner. In order to limit or prevent the damaging effects of inflammation, the immune system is normally well equipped with methods to regulate the balance of pro- and anti-inflammatory cytokines. However, many diseases or disorders will occur when the injured tissue is unable to create this appropriate cytokine balance and interaction (Feghali and Wright, 1997). The onset of the inflammation process is, therefore, not attributable to a single cytokine. For example, an elevation in pro-inflammatory cytokines will not necessarily cause exaggerated inflammation if it is accompanied by an elevation in anti-inflammatory cytokine levels.
Although many cytokines are involved in the inflammation process, some cytokines have a central role in the process and have recently been examined, as possible targets for anti-inflammatory products. For example, the pro-inflammatory cytokine TNF-α has been clearly established as playing a pivotal role in many chronic inflammatory diseases and has been targeted for such therapies as monoclonal antibodies, soluble TNF-α receptors, TNF-converting enzyme, and other anti-TNF-α therapies (Lewis and Manning, 1999).
The anti-inflammatory cytokine IL-10 also plays a critical role in the inflammation process to down-regulate the acute inflammation response. Because of this property, IL-10 has been actively studied as a therapeutic means of controlling inflammation related diseases through gene therapy (Lewis and Manning, 1999; Sacca et al., 1997).
A thylakoid extract that has anti-oxidant properties, as described in the patent publication WO01/49305 has been tested for its capacity as a modulator of cytokines, and in combination with other anti-inflammatory agents. This extract is provided in the form of specific formulations that ensure the integrity and stability thereof. To simplify terminology, the terms “thylakoids”, “thylakoid extract”, and “extract” are used hereinbelow and are meant to cover all the specific formulations comprising thylakoids.
TNF-α and IL-10 have been selected as preferred examples of cytokines that are systematically involved in inflammation, notwithstanding the nature of the causative agent or the nature of the tissue or system.
There is an increasing body of literature suggesting that these two cytokines are involved in the expression of inflammatory diseases and disorders exemplified but not limited to those affecting the following tissues:    Skin: psoriasis (Reich et al., 2001), cutaneous inflammation (Berg et al., 1995), atopic dermatitis (Lee et al., 2000);    Brain: encephalitis (Deckert et al., 2001);    Gastrointestinal tract: inflammatory bowel disease (Gasche et al., 2000), Crohn's disease (Narula et al., 1998), colitis (Moriguchi et al., 1999);    Eye: infected cornea (Yan et al., 2001);    Lung: hypersensitivity pneumonitis (Gudmundsson et al., 1998), chronic lung inflammation (Jones et al., 1996);    Multiorgan: ischemia-reperfusion injury (Daemen et al., 1999);    Autoimmune disease: rheumatoid arthritis (Maini et al., 1997; van Roon et al., 1996); and    Hyper-reactivity: asthma (Thomas, 2001).
The state of the art and the availability of a performing and stabilized thylakoid extract prompted the present inventors to test the extract against IL-10 and/or TNF-α expression.
Besides the capacity of affecting cytokines expression, the complementarity of the thylakoid extract with other anti-inflammatory agents has been investigated.